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Fig. 6. Effects of GSK3
and E-cadherin inhibitors on Ccnd1 and Mmp7 gene expression. (A,B) WT (
) and Timp3–/– (
) MECs were treated with the GSK3 inhibitor, SB-216763 (20 mM for 24 hours) and the E-cadherin blocking antibodies, ECCD-1 (200 mg/ml for 6 hours) and DECMA-1 (0.5 mg/ml for 6 hours). Expression of -catenin target genes, Ccnd1 (A) and Mmp7 (B) was assessed by TaqMan real-time PCR and normalized to 18S RNA values. (A) Inhibition of GSK3 led to an overall increase in Ccnd1 mRNA levels in WT and Timp3–/– MECs, but still showed significantly lower levels in Timp3–/– MECs compared with treated WT MECs. Similarly, treatment with SB-216763 of WT and Timp3–/– MECs generally increased Mmp7 mRNA levels. Upon GSK3 inhibition WT and Timp3–/– expression of Mmp7 became comparable (B). E-cadherin inhibition using two different blocking antibodies decreased Ccnd1 and increased Mmp7 mRNA levels in treated WT MECs but had no effect on Timp3–/– MECs. (C) Treatment with SB-216763, ECCD-1 and DECMA-1 elevated WT MEC levels of dephosphorylated -catenin (de-P--cat) to those of untreated Timp3–/– MEC levels. The -catenin levels in Timp3–/– MECs may be maximal as treatment with the GSK3 inhibitor did not further increase dephosphorylated -catenin levels. ANOVA was performed to assess statistical significance between WT and Timp3–/– cells. Values are expressed as mean ± s.e.m. of three independent experiments.
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